Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats.

The long-term administration of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, produces coronary vascular remodeling and myocardial hypertrophy in animals. This study used a rat model to investigate the role of angiotensin I converting enzyme (ACE) in the pathogenesis of such changes. We studied the following groups, all of which received drug treatment in their drinking water: untreated controls, and those administered L-NAME, L-NAME, and an ACE inhibitor (ACEI), and L-NAME and hydralazine. Cardiovascular structural changes and tissue ACE activities were evaluated after the first, fourth, and eighth week of treatment. In rats treated with L-NAME alone, vascular remodeling was evident at the fourth and eighth week, and myocardial hypertrophy was present at the eighth week of treatment. The vascular and myocardial remodeling were characterized by increased tissue ACE activities and immunodetectable ACE in those tissues. These changes were markedly reduced by ACEI, but not by hydralazine treatment. Increased local ACE expression may thus be important in the pathogenesis of cardiovascular remodeling in this model.

Mechanotransduction of rat aortic vascular smooth muscle cells requires RhoA and intact actin filaments.

The growth-promoting effect of mechanical stress on vascular smooth muscle cells (VSMCs) has been implicated in the progress of vascular disease in hypertension. Extracellular signal-regulated kinases (ERKs) have been implicated in cellular responses, such as vascular remodeling, induced by mechanical stretch. However, it remains to be determined how mechanical stretch activates ERKs. The cytoskeleton seems the most likely candidate for force transmission into the interior of the cell. Therefore, we examined (1) whether the cytoskeleton involves mechanical stretch-induced signaling, (2) whether Rho is activated by stretch, and (3) whether Rho mediates the stretch-induced signaling in rat cultured VSMCs. Mechanical stretch activated ERKs, with a peak response observed at 20 minutes, followed by a significant increase in DNA synthesis. Treatment with the ERK kinase-1 inhibitor, PD98059, inhibited the stretch-induced increase in DNA synthesis. Cytochalasin D, which selectively disrupts the network of actin filaments, markedly inhibited stretch-induced ERK activation. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to mechanical stretch. Botulinum C3 exoenzyme, which inactivates Rho p21 (known to participate in the reorganization of the actin cytoskeleton), attenuated stretch-induced ERK activation. Inhibition of Rho kinase (p160ROCK) also suppressed stretch-induced ERK activation dose dependently. Our results suggest that mechanotransduction in VSMCs is dependent on intact actin filaments, that Rho is activated by stretch, and that Rho/p160ROCK mediates stretch-induced ERK activation and vascular hyperplasia.

Endothelin-1 is not involved in serotonin-induced coronary spasm in a swine model.

OBJECTIVES: The role of endothelin-1 (ET-1) in the pathogenesis of coronary artery spasm is not well understood. We aimed to determine if ET-1 is involved in serotonin-induced coronary spasm in the swine model. METHODS: In 10 miniature pigs, a segment of the left anterior descending coronary artery was denuded and irradiated with X-ray. Three months after endothelial denudation, coronary vasomotion was assessed in vivo by quantitative arteriography. RESULTS: Intracoronary serotonin at 10 micrograms/kg provoked coronary spasm (augmented narrowing of the luminal diameter) at the denuded site (diameter reduction 93 +/- 4%) but not at the non-denuded control site (19 +/- 4%, P < 0.01) associated with ST segment elevation in the region perfused by the denuded artery. Intracoronary administration of ET-1 at 25 ng/kg caused mild vasoconstriction of the denuded (26 +/- 4) and non-denuded site (16 +/- 3%, n.s.), but provoked ST segment elevation in the regions perfused by both the denuded and non-denuded arteries. The treatment with an endothelin antagonist (BQ123 0.1 mg/kg) significantly attenuated coronary vasoconstriction and ST segment elevation evoked with ET-1, but did not alter serotonin-induced vasoconstriction either at the denuded or control site. CONCLUSIONS: The results of this study suggest that endogenous ET-1 may not be involved in the pathogenesis of serotonin-induced coronary spasm in our swine model.

Intracellular signaling in rat cultured vascular smooth muscle cells: roles of nuclear factor-kappaB and p38 mitogen-activated protein kinase on tumor necrosis factor-alpha production.

Lipopolysaccharide (LPS) is responsible for initiating host responses leading to septic shock, and tumor necrosis factor-alpha (TNF alpha) is thought to be its primary mediator. In addition, TNF alpha is one of the major components of the pathogenesis of insulin resistance in various conditions. It has been shown that LPS induced TNF alpha production in rat vascular smooth muscle cells (VSMC). However, little is known about the signaling pathway by which VSMC in culture produce TNF alpha. We investigated the possible signaling components involved in this pathway. LPS elicited phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) and p38 MAPK, degradation of inhibitor of kappaB (IkappaB), and an increase in nuclear binding activity of activating protein-1 and nuclear factor-kappaB (NF-kappaB). Different types of NF-kappaB inhibitors, pyrrolidine dithiocarbamate and MG132, which specifically abolished IkappaB degradation and subsequent NF-kappaB activation by LPS, suppressed TNF alpha secretion from VSMC. Although PD98059, a specific MAPK kinase inhibitor and SB203580, a specific p38 MAPK inhibitor, had no effect on NF-kappaB activity, SB203580 suppressed TNF alpha secretion; however, PD98059 did not. A cotransfection assay showed that transfection of dominant negative IkappaB or pretreatment with SB203580 suppressed the TNF alpha gene promotor-dependent transcription. TNF alpha messenger RNA expression induced by LPS was inhibited by pyrrolidine dithiocarbamate, MG132, and SB203580, but not by PD98059. These observations indicate that TNF alpha production in VSMC is stimulated by LPS, and its transcription and translation are dependent on NF-kappaB activation through proteasome-mediated IkappaB degradation. It is likely that p38 MAPK may play a critical role in regulating transcription of the TNF alpha gene in VSMC, unlike in other cell lines.

Induction of cyclooxygenase-2 by angiotensin II in cultured rat vascular smooth muscle cells.

Angiotensin II (Ang II) stimulates the release of prostaglandins (PGs) in various cells and tissues. Recently, cyclooxygenase-2 (COX-2) emerged as a new key regulator for PG synthesis. In the present study, we investigated whether Ang II regulates COX-2 expression in cultured rat vascular smooth muscle cells (VSMCs). Ang II markedly increased the expression of COX-2 mRNA in a time- and dose-dependent manner. This effect was completely blocked by the Ang II type 1 receptor antagonist losartan but not by the Ang II type 2 receptor antagonist PD123319. The p42/44 mitogen-activated protein kinase (MAPK) kinase-1 inhibitor PD98059 and the p38 MAPK inhibitor SB203580 significantly suppressed Ang II-induced COX-2 mRNA and protein expression. Ang II did not increase transcription of the COX-2 gene, as examined with a COX-2 promoter/luciferase chimeric plasmid construct. Instead, it suppressed the degradation of COX-2 mRNA. PD98059 and SB203580 markedly enhanced the decay of COX-2 mRNA induced by Ang II, implying that p42/44 and p38 MAPK activated by Ang II play a role in the regulation of COX-2 through stabilization of its mRNA. The COX-2-specific inhibitor NS-398 attenuated Ang II-stimulated DNA and protein synthesis, as well as PGE(2) production by VSMCs. These results suggest that Ang II regulates COX-2 expression and PG production and modulates cell proliferation through MAPK-mediated signaling pathways in rat VSMCs.

Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling in rats.

The aim of the present study was to investigate the effects of long-term blockade of nitric oxide synthesis with the L-arginine analogue N omega-nitro-L-arginine methyl ester (L-NAME) for 8 weeks on coronary vascular and myocardial structural changes. Four groups of Wistar-Kyoto rats were studied: those with no treatment, those treated with L-NAME 1 g/L (3.7 mmol/L in drinking water), those treated with L-NAME 0.1 g/L (0.37 mmol/L in drinking water), and those treated with L-NAME 1.0 g/L and hydralazine 120 mg/L (0.6 mmol/L in drinking water). After 8 weeks, the heart was excised, and the degrees of structural changes in coronary arteries (wall-to-lumen ratio and perivascular fibrosis), myocardial fibrosis, and myocyte size were quantified by an image analyzer. Chronic inhibition of nitric oxide synthesis increased arterial pressure compared with control animals. Chronic inhibition of nitric oxide synthesis caused significant microvascular remodeling (increased wall-to-lumen ratio and perivascular fibrosis). Cardiac hypertrophy was also observed after chronic inhibition of nitric oxide synthesis. Coadministration of hydralazine prevented arterial hypertension but did not affect microvascular remodeling and cardiac hypertrophy induced by the chronic inhibition of nitric oxide synthesis. In addition, chronic inhibition of nitric oxide synthesis caused scattered lesions of myocardial fibrosis, which was significantly attenuated by cotreatment with hydralazine. These results suggest that long-term blockade of nitric oxide synthesis caused coronary microvascular remodeling and cardiac hypertrophy in rats in vivo by a mechanism other than arterial hypertension. In contrast, arterial hypertension contributed to the development of myocardial fibrosis induced by long-term blockade of nitric oxide synthesis.

Involvement of Rho-kinase in angiotensin II-induced hypertrophy of rat vascular smooth muscle cells.

Angiotensin II (Ang II) is now believed to play a critical role in the pathogenesis of hypertrophy and/or hyperplasia of vascular smooth muscle cells (VSMCs). Several G(i)- and G(q)-coupled receptors, including the Ang II type 1 (AT(1)) receptor, activate Rho and Rho-associated kinase in Swiss 3T3 cells and cardiac myocytes. However, little is known about the role of Rho-kinase in Ang II-induced vascular hypertrophy in VSMCs. In the present study, we explored the role of Rho and Rho-kinase in Ang II-induced protein synthesis in VSMCs. In unstimulated cells, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to Ang II (100 nmol/L). This effect was completely blocked by the AT(1) receptor blocker candesartan but not by the Ang II type 2 (AT(2)) receptor antagonist PD123319. Botulinum C(3) exoenzyme, which inactivated RhoA, attenuated Ang II-induced [(3)H]leucine incorporation. The specific Rho-kinase inhibitor, Y-27632, dose-dependently abolished Ang II-induced protein synthesis and also suppressed Ang II-induced c-fos mRNA expression. On the other hand, Y-27632 had no effect on Ang II-stimulated phosphorylation of p70 S6 kinase and extracellular signal-regulated kinase 1/2, which are reported to be involved in Ang II-induced protein synthesis, nor had it any effect on the Ang II-induced phosphorylation of PHAS-I, a heat- and acid-stable eIF-4E-binding protein. The phosphorylation of PHAS-I is regulating for translation initiation. These observations suggest that the Rho, Rho-kinase, and c-fos pathways may play a role in Ang II-induced hypertrophic changes of VSMCs through a novel pathway.

Lysophosphatidylcholine stimulates MAP kinase activity in rat vascular smooth muscle cells.

Lysophosphatidylcholine (lyso-PC) has been implicated in atherogenesis and the inflammatory process. Although lyso-PC has been reported to contribute to the mitogenic effect of oxidized LDL on rat cultured vascular smooth muscle cells (VSMCs), the signaling mechanisms by which lyso-PC promotes its proliferation are poorly characterized. Mitogen-activated protein (MAP) kinases are important mediators involved in the intracellular network of interacting proteins that transduces extracellular cues to intracellular responses. We therefore examined the effect of lyso-PC on MAP kinase activation, proto-oncogene expression, and AP-1 binding activity using cultured rat VSMC. Marked activation of MAP kinase occurred within 10 minutes of lyso-PC treatment, whereupon rapid inactivation ensued. MAP kinase activation by lyso-PC was concentration-dependent (6.25 to 25 micromol/L). Pertussis toxin treatment did not affect lyso-PC-induced MAP kinase phosphorylation. Lyso-PC (25 micromol/L) also increased the mRNA expression of c-fos and c-jun genes. An electrophoretic mobility shift assay showed that AP-1 binding activity was enhanced by lyso-PC. To examine the upstream signaling of MAP kinase, we used several inhibitors on MAP kinase activation induced by lyso-PC. Although lyso-PC induced sustained increase in intracellular Ca2+ concentration, EGTA had no effect on MAP kinase activation induced by lyso-PC. However, protein kinase C inhibitor GF109203X and downregulation of protein kinase C activity by prolonged treatment with phorbol ester inhibited lyso-PC-induced MAP kinase activation. These data suggest that lyso-PC transmits its mitogenic activity through a MAP kinase-AP-1 pathway, which exists downstream of its protein kinase C activation in VSMCs.

Involvement of PYK2 in angiotensin II signaling of vascular smooth muscle cells.

-PYK2, a recently identified Ca2+-sensitive tyrosine kinase, has been implicated in extracellular signal-regulated kinase (ERK) activation via several G protein-coupled receptors. We have reported that angiotensin II (Ang II) induces Ca2+-dependent transactivation of the epidermal growth factor receptor (EGFR) which serves as a scaffold for preactivated c-Src and downstream adaptors (Shc/Grb2), leading to ERK activation in cultured rat vascular smooth muscle cells (VSMC). Herein we demonstrate the involvement of PYK2 in this cascade. Ang II rapidly induced tyrosine phosphorylation of PYK2, whose effect was completely inhibited by an AT1 receptor antagonist and an intracellular Ca2+ chelator. A Ca2+ ionophore also induced PYK2 tyrosine phosphorylation to a level comparable with that by Ang II, whereas phorbol ester-induced phosphorylation was less than that by Ang II. Moreover, PYK2 formed a complex coprecipitable with catalytically active c-Src after Ang II stimulation. Although a selective EGFR kinase inhibitor completely abolished Ang II-induced recruitment of Grb2 to EGFR and markedly attenuated Ang II-induced ERK activation, it had no effect on Ang II-induced PYK2 tyrosine phosphorylation or its association with c-Src and Grb2. These data suggest that the AT1 receptor uses Ca2+-dependent PYK2 to activate c-Src, thereby leading to EGFR transactivation, which preponderantly recruits Grb2 in rat VSMC.

Coronary vascular ATP-sensitive potassium channels are activated to a greater extent in spontaneously hypertensive rats than in Wistar-Kyoto rats.

OBJECTIVE: To determine whether opening of coronary vascular adenosine 5'-triphosphate (ATP)-sensitive potassium (KATP) channels is involved in the maintenance of resting coronary flow in hypertrophied hearts. METHODS: We examined the effects of glibenclamide, a selective inhibitor of KATP channels, on basal coronary vascular tone in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Using a Langendorff system, the hearts from WKY rats and SHRs were isolated and perfused with oxygenated Krebs-Henseleit solution at a constant perfusion pressure of 60 and 100 mmHg respectively. RESULTS: Basal coronary flow and myocardial oxygen consumption (MV02) were similar in SHRs and WKY rats. The percentage decreases in coronary flow with glibenclamide at graded doses were greater (P < 0.01) in SHRs than in WKY rats (n = 8), whereas the percentage decreases in MV02 with glibenclamide were similar in the two groups. The decreases in coronary flow caused by U46619 (a thromboxane A2-mimetic agent) were similar in SHRs and WKY rats (n = 4). The increase in coronary flow caused by pinacidil (a KATP opener) was greater in SHRs than in WKY rats; glibenclamide prevented the pinacidil-induced increase in coronary flow in both SHRs and WKY rats. There was a significant positive correlation between the glibenclamide-induced decrease in coronary flow and the degree of left ventricular hypertrophy (r = 0.54, P < 0.05). CONCLUSION: Our results suggest that the basal opening state of coronary vascular KATP channels is activated to a greater extent in SHRs than WKY rats, which may contribute to the maintenance of basal myocardial perfusion in hypertrophied hearts.

Epidermal growth factor receptor is indispensable for c-Fos expression and protein synthesis by angiotensin II.

We have reported that angiotensin II induces the epidermal growth factor (EGF) receptor transactivation leading to extracellular signal-regulated kinase (ERK) activation in rat vascular smooth muscle cells. Here, we report that the EGF receptor kinase inhibitor AG1478 and the ERK kinase inhibitor PD98059 markedly inhibited angiotensin II-induced c-Fos expression and protein synthesis but not c-Jun expression in these cells. These data suggest that the EGF receptor transactivation and subsequent ERK activation are indispensable for angiotensin II-mediated growth promotion of vascular smooth muscle cells providing a new mechanistic insight whereby angiotensin II contributes abnormal vascular remodeling.

The effect of dexamethasone on electroencephalograms in patients with brain tumors with specific reference to topographic computer display of delta activity.

Nine patients with primary or metastatic brain tumors were examined with topographic computer display of delta activity before and after administration of dexamethasone. In six cases with primary malignant or metastatic brain tumors a diminution of delta distribution was observed following intravenous administration of dexamethasone, and no significant changes of the topographic map of the delta wave were obtained in two cases of benign brain tumor without clinical signs of increased intracranial pressure. Some of the changes of delta activities are believed to be correlated with the amount of peritumor edema. It is noteworthy that the change of focal delta activities by steroid was detected 20 minutes after administration.

Effects of N-nitro-L-arginine on coronary artery tone and reactive hyperemia after brief coronary occlusion in conscious dogs.

AIM: To determine the role of an endothelium-derived relaxing factor (nitric oxide) in controlling basal coronary tone and coronary vasomotion after brief coronary occlusion (reactive hyperemia). METHODS: In 10 chronically instrumented conscious dogs, we studied the diameter changes of the large epicardial coronary artery and coronary blood flow in response to intracoronary administration of acetylcholine (0.1 and 1 microgram) and brief coronary occlusion for 5 and 20 s before and after intracoronary infusion of N-nitro-L-arginine (LNNA). RESULTS: Intracoronary infusion of LNNA (1, 3, and 10 mg) decreased the diameter of the large epicardial coronary artery and coronary blood flow in a dose-dependent manner without altering arterial pressure and heart rate. LNNA (10 mg) significantly attenuated the increase in artery diameter and coronary blood flow by acetylcholine. The ratio of artery dilation to the blood flow response after acetylcholine was not affected by LNNA. LNNA (10 mg) significantly decreased the ratio of repayment to debt flow volume of reactive hyperemia, but did not affect the ratio of peak to resting flow; it also significantly attenuated the reactive dilation of the large epicardial coronary artery after reactive hyperemia. The ratio of artery dilation to repayment flow volume (micron/ml) during reactive hyperemia was attenuated significantly by LNNA. CONCLUSION: These findings suggest that endothelium-derived nitric oxide may contribute to basal coronary tone and that reactive dilation of the large epicardial coronary artery during reactive hyperemia was caused by flow-mediated nitric oxide release, whereas coronary artery dilation after acetylcholine was caused largely by the direct receptor-mediated release of nitric oxide.

Idiopathic cerebral hemorrhage during delivery in normotensive women--case reports.

Two normotensive pregnant women who developed cerebral hemorrhage during delivery are reported. The hematoma was small and subcortical in one and large and putaminal in the other. They had no history of hypertension, cerebrovascular disorders, or eclampsia. Cerebral angiography studied in one case revealed no abnormalities. Idiopathic cerebral hemorrhage during delivery without any evidence of vascular risk factors is discussed.

Radiotherapy of bile duct carcinoma using intracatheter 198Au grains.

Eight patients with bile duct carcinoma were treated with radiotherapy using intracatheter 198Au grains. The intracatheter 198Au grains in an inner tube were inserted into a percutaneous transhepatic catheter. A plastic tip was placed between these grains to improve spatial and temporal dose allocation. This method and 192Ir wire irradiation resemble each other closely in dose distribution, but the former has the following advantages over the latter. 1) The number of 198Au grains used can be changed quite easily in accordance with the length of the stenosis. 2) The half-life of 198Au is about 2.7 days, and a dose of 25-40 Gy at 1.0 cm from the source is delivered over this period. The medical staff can protect themselves from radioactivity when the sources are withdrawn after brachytherapy. 3) 192Ir wire is not used very frequently in spite of its long half-life (74 days) because bile duct carcinoma is uncommon. 4) In Japan, 198Au grains can be purchased on a weekly basis, so treatment plans can be easily made. The eight patients also received external irradiation and the median survival after onset of radiotherapy was 7.9 months. There have been few systemic or local complications.

Immediate relief of spontaneous coronary artery spasm by intracoronary infusion of an endothelium-dependent vasodilator, substance P.

This report describes a 45-year-old Japanese man who had episodes of anginal chest pain on effort. Coronary arteriography in the baseline state revealed subtotal occlusion in the mid-portion of the left anterior descending coronary artery. After intracoronary infusion of an endothelium-dependent vasodilator, substance P, the subtotal occlusion was immediately abolished. We concluded that endothelium-dependent vasodilation evoked with substance P was present at the site where coronary vasospasm occurred spontaneously in our case.

[Effectiveness of radiation therapy in metastatic bone tumors].

The effectiveness of radiation therapy for metastatic bone tumor in 61 patients with 80 lesions has been evaluated. Relief from pain resulted in 87% all the treated lesions and this continued for more than two years in eight lesions of seven patients. There was no statistical difference in the pain relief achieved among the sites and the histologies of the primary tumors, and also no difference was noted as to the treated regions. Pain relief from the lesions of a squamous cell carcinoma tended to begin with smaller doses when compared with that of an adenocarcinoma. Our results suggest that radiation therapy in general appears to be an effective technique in achieving long term control of pain, but for some patients those with a generalized metastases or an uncontrolled primary tumor, for example, this tedious treatment has limited value and so should be withheld.

PKC inhibitors prevent endothelial dysfunction after myocardial ischemia-reperfusion in rats.

The intracellular mechanism for endothelial dysfunction after myocardial ischemia-reperfusion remains to be elucidated. It has been reported that activation of protein kinase C (PKC) occurs after myocardial ischemia-reperfusion and that the activation impairs endothelium-dependent relaxation. Thus we examined the role of PKC activation in the ischemia-reperfusion-induced endothelial dysfunction. Isolated rat hearts perfused with a constant flow were subjected to global ischemia for 15 min followed by reperfusion for 20 min. Coronary vascular responses to the endothelium-dependent vasodilators acetylcholine (ACh) and bradykinin (BK) and the endothelium-independent vasodilator sodium nitroprusside (SNP) were examined before and after the ischemia-reperfusion. Endothelium-dependent relaxations to ACh and BK were impaired after the ischemia-reperfusion, whereas endothelium-independent relaxations to SNP were unaffected. Pretreatment with a PKC inhibitor, staurosporine, H7, or calphostin C, prevented the impairments. Phorbol 12-myristate 13-acetate, a PKC-activating phorbol ester, attenuated the relaxations to ACh and BK but not those to SNP. These results suggest that PKC activation may be involved in part in the ischemia-reperfusion-induced endothelial dysfunction.